The present invention relates to detection of specific sequences contained in DNAs of test subjects, detection of polymorphisms of genes, analysis of single nucleotide polymorphisms (SNPs), and the like.
The progress of the human genome mapping project is prompting a trend towards applying DNA sequence information to medical diagnoses and various industries, for example, in producing new drugs. Particularly, in a medical field, noticeable is a trend towards revealing and applying gene functions. In particular, analyses of gene expression profiles, in which genes active in a body are examined, and analyses of single nucleotide polymorphisms (SNPs) in genomes, which may be a cause of variations in gene expression, are drawing attention.
In genomes, single nucleotide polymorphisms are said to be present approximately one per 1000 bases, which means every individual has a huge number of single nucleotide polymorphisms. These single nucleotide polymorphisms are considered to be associated with characteristics of individuals so that the analysis of these single nucleotide polymorphisms is expected to provide a therapy guide or the like for the treatment of diseases for the individuals.
The number of single nucleotide polymorphisms present in genomes of one human being is enormous, and the type of single nucleotide polymorphisms in genomes of many people are polymorphic, in which two kinds of bases appear in particular sites. It is necessary to examine single nucleotide polymorphisms occurring at sites of mutation and further to reveal their correlation with diseases or the like, which requires the accumulation of enormous amounts of DNA data. Thus, development of appropriate methods to meet this requirement is in need.
At present, various methods for analyzing base substitutions are available. They are generally divided into two types, i.e., methods for detecting unknown base substitutions and methods for examining, for example, whether certain known base substitutions are found in many people or whether these known substitutions have causal relationships with certain diseases.
Examples of the methods for detecting unknown base substitutions include a method in which a base sequence is determined using a DNA sequencer, and a method in which hybridization of a DNA probe and a target DNA is detected using a DNA probe array (DNA chip or gene chip).
On the other hand, in order to detect single nucleotide polymorphisms at known positions, methods, such as an SSCP (single strand configuration polymorphism) method, an invader assay (Nature Biotechnology, 17, 292-296 (1999)), and DASH (Nature Biotechnology, 17, 87-88 (1999)), have been developed and implemented for practical use. All of these methods use laser or the like as a light source.
In synthesizing a DNA complementary strand using a primer, complementary strand extension may or may not proceed depending on whether the 3xe2x80x2-end of the primer is complementary to the target or not. Accordingly, a method, in which a PCR amplification is performed using a primer having the 3xe2x80x2-end at an accordant position of mutation and products are analyzed using gel electrophoresis or the like (ARMS: amplified refractory mutation system; Nucleic Acids Research, 17, 2503-2515 (1989)), has also been used. In this case, a key point is how exclusively the complementary strand synthesis occurs in the presence of a target of interest.
In the SSCP method, single, nucleotide polymorphisms can be detected based on the observation that the configurations of single-stranded DNAs with and without mutation during electrophoresis are different, and these configurations influence electrophoretic mobility.
In the invader assay, a triple-stranded chain is formed with a DNA probe of a DNA probe array, a target DNA and a DNA probe, and whether a part of the probe is cleaved with an enzyme is detected.
In DASH, a target DNA and a probe DNA are hybridized under the presence of an intercalator, and light generated from the intercalator by irradiation is observed while changing a temperature. The double stranded chain is dissociated and the emission ceases if mutation is present in the target DNA.
On the other hand, anew technique is about to be implemented, in which a short DNA base sequence is determined in a short time and then mutations are examined using the determined DNA base sequence.
For example, Nyren et al. have proposed a method for determining a DNA base sequence (pyrosequencing), in which a target DNA is hybridized with a primer, pyrophosphate produced by a complementary strand extension reaction is converted into ATP, the ATP is reacted with luciferin to emit light, and this chemiluminescence is detected to discriminate the substrate (dNTP) incorporated during the complementary strand extension reaction. The pyrosequencing has drawn an attention as a simple and easy method for determining a DNA sequence without the need for gel electrophoresis. Recently, detection of SNPs using this pyrosequencing has been reported (Anal Biochemistry, 280, 103-110 (2000)). This method does not require the use of a new light source because it utilizes chemiluminescence.
Mutations which appear in genome DNAs are associated with characteristics of organisms, sensitivity to diseases and medicines, and the like, and thus a huge number of subjects have to be analyzed. Since individuals to be tested are numerous, there is a demand for a method and an apparatus which suit for a simple and large scale operation at a low running cost.
Further, SNP measurements are necessary for individual genome samples, as well as for samples from a patient group and a healthy subject group for comparison. Namely, in order to study a causative relationship between particular SNPs and a certain disease it is necessary to compare the incidence of the SNPs (allele frequency) in the two groups. This comparison study can be most efficiently and easily carried out by combining DNAs of each group and quantitatively analyzing the SNPs contained in the combined DNAs. However, a quantitative method accurate enough for such analyses remains to be developed. Further problems arise upon measurement of SNPs even with individual samples, as described below.
In various methods which have been proposed previously, a targeted DNA region is amplified by PCR and the resulting DNA fragments are used as a target. Accordingly, the use of PCR is inevitable.
In the DASH, it is necessary to use a detector equipped with a laser light source and a filter to remove scattering light, which requires a large-scale apparatus.
In a pyrosequencing for detecting chemiluminescence, pyrophosphate is converted into ATP to generate chemiluminescence and the resulting photo-emission is detected. However, the pyrosequencing has disadvantages such that four kinds of dNTP (dATP, dTTP, dGTP and dCTP) have to be added independently in a designated order to a reaction part, that the photo-emission intensity is weak, that a system for dNTP injection is necessary, and that a considerably large-scale apparatus is required.
Further problems in this method are that because reagents are injected in a designated order, the time required for the analysis is 10-20 minutes although it is shorter than that with the use of electrophoresis, and that sample DNAs have to be PCR-amplified to increase the number of DNA copies of the target region because the method is not sensitive enough without amplification.
Furthermore, in pyrosequencing, if a multiple number of base substitutions exist in a sample to be tested, the resulting spectrum may be too complicated to perform accurate analyses.
In order to apply the pyrosequencing for the detection of DNA mutations, it is important to further improve the sensitivity so that the DNA mutations can be readily detected and DNA strands containing multiple kinds of mutations can be analyzed.
An object of the present invention is to provide a tool necessary to create a data base applicable for genetic diagnoses, new drug manufacturing using genes, and gene therapies, and further to provide a method, an apparatus, test reagents and the like for examining gene DNAs.
More specifically, an object of the present invention is to provide a highly sensitive, easy method and an apparatus for detecting DNA mutations, in which the use of excitatory light source is not necessary and the reaction is simple and quick, and further to provide a method and an apparatus for detecting DNA mutations, in which a sample containing multiple mutations can be analyzed at a high reliability.
Another object of the present invention is to provide a technique for readily screening interrelations between a certain disease and SNPs.
In the present invention, in order to examine the presence or absence of a target DNA of interest, or the presence or absence of mutations, a DNA probe (primer) capable of being engaged in complementary strand extension is hybridized with the target DNA, pyrophosphate generated as a result of the complementary strand extension reaction is converted into ATP to emit light, and this photo-emission is detected. When the target DNA sequence is not present in the test sample, the complementary strand extension does not take place so that no photo-emission is observed.
Further, in the present invention, probes are prepared to have the 3xe2x80x2-end base at a position accordant to mutations and have different 3xe2x80x2-end sequences so that the complementary strand extension reaction occurs only if mutation exists or the complementary strand extension reaction does not occur only when mutation exists. Thus, this method can be used also for detecting mutations. This is because the complementary strand extension reaction starting from the primer highly depends on whether the hybridization matching at the 3xe2x80x2-ends is perfect or imperfect (Kwok S. et al., Nucleic Acids Res., 18, 999-1005 (1990); Huang M. M., Arnhein N., Goodman M. R., Nucleic Acids Res., 20, 4567-4573 (1992)).
In order to accurately switch on/off of the primer complementary strand extension reaction in response to a DNA template for targeted SNP measurements, an artificial mismatch is introduced near the 3xe2x80x2-end of the primer. The presence or absence of the complementary strand synthesis is examined by converting the produced pyrophosphate into ATP, reacting the ATP with a chemiluminescent reagent and optically detecting the resulting chemiluminescence. In order to accomplish a highly accurate detection by this method, pyrophosphate contained as an impurity and pyrophosphate resulting from heat decomposition of nucleic acid substrates, which generate a background light, are minimized as much as possible, and at the same time, the chemiluminescence resulting from the complementary strand extension is detected using two primers having the end bases complementary to a wild type and a mutant.
In a conventional pyrosequencing, photo-emission intensity is weak because the photo-emission uses pyrophosphate resulting from a complementary strand extension reaction with only one base. Contrastingly in the present invention, the complementary strand extension reaction proceeds with more than dozens of bases and chemiluminescence is detected by converting the resulting pyrophosphate into ATP so that the photo-emission intensity is more than two orders stronger than that attained by the conventional pyrosequencing.
Further, it is possible to enhance the photo-emission intensity by repeating the decomposition of the double-stranded chain resulting from the complementary strand extension reaction using an enzyme and the process of the complementary strand extension. Thus, the presence or absence of specific sequences or mutations, and the ratio of mutation contained in combined genome samples can be detected without PCR-amplification of the samples.
In the present invention, substrates for the complementary strand synthesis, i.e., dNTPs, are added in a controlled manner upon the complementary strand extension starting from a primer, pyrophosphate produced in response to the complementary strand extension reaction is detected by chemiluminescence emission, and thus the presence or absence of base mutations and the presence of a wild type and a mutant in a target DNA are detected. The presence or absence of substitution of base sequences or base mutation can be examined by a simple and highly sensitive method using primers designed such that their 3xe2x80x2-ends come to the position of the anticipated base substitution in the target DNA and the complementary strand extension may or may not occur depending on the presence or absence of the substitution.
When the complementary strand extension occurs, multiple dNTPs are simultaneously added to proceed the complementary strand extension all the way through and produce a large amount of pyrophosphate so that the intensity of chemiluminescence can be enhanced to attain a highly sensitive detection of base mutations.
Multiple primers are prepared taking the sequence of a target DNA into consideration, and the progress of complementary strand extensions starting from these various primers are detected to discriminate the presence of multiple kinds of mutations. Further, multiple-kinds of base substitutions can be analyzed with high reliability by sorting and holding multiple primers for the reaction according to known standard DNA sequences. When a sample contains various mutants, multiple primers are prepared referring to the standard base sequence and immobilized on a solid surface by kind, and complementary strand extension reactions are carried out using these primers to monitor the presence of mutants.
An analysis of test samples containing various mutations has been extremely difficult with conventional methods. However, the mutations can be detected using a simple method and an apparatus according to the present invention.